Biosynthesis of the mono-hydroxy "HETEs" (hydroxyeicosatetraenoic acids) is widespread, yet highly tissue-specific in terms of mechanism of synthesis and structure of the products. A lack of understanding of the mechanism of cytochrome P-450-catalyzed HETE synthesis is a fundamental gap in our knowledge that we aim to address. A second facet of this project involves study of "R" lipoxygenases, both to compare their primary structures to the better known "S" specific enzymes and to facilitate investigation of their unique catalytic activities. 1. P-450-dependent HETE synthesis will be studied primarily in rat liver microsomes. Fatty acid analogues will be used to probe the substrate specificity. The mechanism will be examined by (i) use of an oxygen surrogate (iodosylbenzene) that can support only mono-oxygenations, (ii) by identification of potential "side-products" that might distinguish the P-450 mechanism, (iii) by measurement of the stereospecificity of the C- 10 hydrogen abstraction associated with the prominent 12R-oxygenation, (iv) by detection of possible intermediates in HETE synthesis, (v) by comparison with model heme systems, and (vi) by characterization of the HETE synthesis by selected P-450 isozymes. Using this information we will develop methods that discriminate P-450-dependent HETE synthesis and apply the methods in tissues (psoriatic, corneal) that may form HETEs by this pathway. 2. We will use a molecular approach to clone and sequence a marine "R" lipoxygenase. We will then use the molecular probes and sequences to clone additional "R" lipoxygenases. Expression of the "R" enzymes will permit an analysis of the unique catalytic activities and of the basis of the "R" versus "S" stereospecificity. By comparison with the more fully characterized "S" lipoxygenases, the results will afford a new insight into structure-function characteristics of the lipoxygenase family of dioxygenases.